U.S. patent application number 10/727010 was filed with the patent office on 2004-12-09 for methods for selecting and screening for transformants.
This patent application is currently assigned to ProdiGene, Inc.. Invention is credited to Howard, John A., Pinkerton, T. Scott, Wild, James R..
Application Number | 20040250298 10/727010 |
Document ID | / |
Family ID | 32850726 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040250298 |
Kind Code |
A1 |
Pinkerton, T. Scott ; et
al. |
December 9, 2004 |
Methods for selecting and screening for transformants
Abstract
The present invention provides novel methods for determining
whether a cell has incorporated a polynucleotide comprising the use
of organophosphate hydrolase activity as a marker which has both
selectable and screenable properties.
Inventors: |
Pinkerton, T. Scott;
(College Station, TX) ; Howard, John A.; (College
Station, TX) ; Wild, James R.; (College Station,
TX) |
Correspondence
Address: |
MCKEE, VOORHEES & SEASE, P.L.C.
801 GRAND AVENUE
SUITE 3200
DES MOINES
IA
50309-2721
US
|
Assignee: |
ProdiGene, Inc.
College Station
TX
|
Family ID: |
32850726 |
Appl. No.: |
10/727010 |
Filed: |
December 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60430626 |
Dec 3, 2002 |
|
|
|
Current U.S.
Class: |
800/3 ;
800/278 |
Current CPC
Class: |
G01N 33/5005
20130101 |
Class at
Publication: |
800/003 ;
800/278 |
International
Class: |
G01N 033/00; C12N
015/87; C12N 015/80; C12N 015/85 |
Claims
We claim:
1. A method for determining whether a cell has incorporated and
expresses a polynucleotide, the method comprising: introducing to a
cell a construct comprising a polynucleotide encoding an enzyme
having organophosphate hydrolase activity; contacting the cell with
an organophosphate such that if the cell does not contain the
construct and an enzyme having organophosphate hydrolase activity
is not thereby produced, the cell growth is inhibited, thereby
determining whether the cell has incorporated the
polynucleotide.
2. The method of claim 1, wherein the organophosphate is selected
from the group consisting of acephate, azinophos-methyl,
demeton-S-methyl, malathion, phosalone, amiprophos-methyl,
bensulide, butamiphos, piperophos, paraoxon, DFP, coumaphos, soman,
and VX.
3. The method of claim 1, wherein the organophosphate is
bensulide.
4. The method of claim 1, wherein the polynucleotide is SEQ ID
NO:1.
5. The method of claim 1, wherein the cell is selected from the
group consisting of a plant cell, an animal cell, and a fungal
cell.
6. The method of claim 5, wherein the plant cell is a maize plant
cell.
7. A method for determining whether a cell has incorporated and
expresses a polynucleotide, the method comprising: introducing to a
cell a construct comprising a polynucleotide encoding an enzyme
having organophosphate hydrolase activity; contacting the cell with
an organophosphate such that if the cell contains the construct and
an enzyme having organophosphate hydrolase activity is thereby
expressed, the enzyme having organophosphate hydrolase activity
hydrolyzes the organophosphate; and detecting the hydrolysis,
thereby determining whether the cell has incorporated a
polynucleotide.
8. The method of claim 7, wherein the organophosphate is selected
from the group consisting of acephate, azinophos-methyl,
demeton-S-methyl, malathion, phosalone, amiprophos-methyl,
bensulide, butamiphos, piperophos, paraoxon, DFP, coumaphos, soman,
and VX.
9. The method of claim 7, wherein the organophosphate is
amiprophos-methyl.
10. The method of claim 7, wherein the polynucleotide is SEQ ID
NO:1.
11. The method of claim 7, wherein the cell is selected from the
group consisting of a plant cell, an animal cell, a bacterial cell,
and a fungal cell.
12. The method of claim 11, wherein the plant cell is a maize plant
cell.
13. A method for determining whether a cell has incorporated a
first polynucleotide, the method comprising: introducing to a cell
a construct comprising a first polynucleotide and a second
polynucleotide, wherein the second polynucleotide encodes an enzyme
having organophosphate hydrolase activity; contacting the cell with
an organophosphate such that if the cell does not contain the
construct and an enzyme having organophosphate hydrolase activity
is not thereby produced, the cell growth is inhibited, thereby
determining whether the cell has incorporated a first
polynucleotide.
14. The method of claim 13, wherein the organophosphate is selected
from the group consisting of acephate, azinophos-methyl,
demeton-S-methyl, malathion, phosalone, amiprophos-methyl,
bensulide, butamiphos, piperophos, paraoxon, DFP, coumaphos, soman,
and VX.
15. The method of claim 13, wherein the organophosphate is
bensulide.
16. The method of claim 13, wherein the second polynucleotide is
SEQ ID NO:1.
17. The method of claim 13, wherein the cell is selected from the
group consisting of a plant cell, an animal cell, and a fungal
cell.
18. The method of claim 17, wherein the plant cell is a maize plant
cell.
19. A method for determining whether a cell has incorporated a
first polynucleotide, the method comprising: introducing to a cell
a construct comprising a first polynucleotide and a second
polynucleotide, wherein the second polynucleotide encodes an enzyme
having organophosphate hydrolase activity; contacting the cell with
an organophosphate such that if the cell contains the construct and
an enzyme having organophosphate hydrolase activity is thereby
expressed, the enzyme having organophosphate hydrolase activity
hydrolyzes the organophosphate; and detecting the hydrolysis,
thereby determining whether the cell has incorporated a first
polynucleotide.
20. The method of claim 19, wherein the organophosphate is selected
from the group consisting of acephate, azinophos-methyl,
demeton-S-methyl, malathion, phosalone, amiprophos-methyl,
bensulide, butamiphos, piperophos, paraoxon, DFP, coumaphos, soman,
and VX.
21. The method of claim 19, wherein the organophosphate is
amiprophos-methyl.
22. The method of claim 19, wherein the second polynucleotide is
SEQ ID NO:1.
23. The method of claim 19, wherein the cell is selected from the
group consisting of a plant cell, an animal cell, a bacterial cell,
and a fungal cell.
24. The method of claim 23, wherein the plant cell is a maize plant
cell.
25. The method of claim 1, wherein the cell does not naturally
contain an enzyme having significant organophosphate hydrolase
activity, and wherein the cell is a plant cell.
26. The method of claim 25, wherein the plant cell is a maize plant
cell.
27. The method of claim 7, wherein the cell does not naturally
contain an enzyme having significant organophosphate hydrolase
activity, and wherein the cell is a plant cell.
28. The method of claim 27, wherein the plant cell is a maize plant
cell.
29. The method of claim 13, wherein the cell is a plant cell.
30. The method of claim 29, wherein the plant cell is a maize plant
cell.
31. The method of claim 19, wherein the cell is a plant cell.
32. The method of claim 31, wherein the plant cell is a maize plant
cell.
33. A method for determining the presence of absence of a
polynucleotide in a cell comprising: introducing into a cell an
expression construct comprising a polynucleotide as set forth in
SEQ ID NO:1, wherein said polynucleotide encodes an enzyme having
organophosphate hydrolase activity; contacting said cell with an
organophosphate; and analyzing for the presence of hydrolysis
detected by a method selected from spectrophotometry, fluorescence
and phosphorescence.
34. The method of claim 33, wherein the organophosphate is selected
from the group consisting of acephate, azinophos-methyl,
demeton-S-methyl, malathion, phosalone, amiprophos-methyl,
bensulide, butamiphos, piperophos, paraoxon, DFP, coumaphos, soman,
and VX.
35. The method of claim 33, wherein the organophosphate is
amiprophos-methyl.
36. The method of claim 33, wherein the cell is selected from the
group consisting of a plant cell, an animal cell, a bacterial cell
and a fungal cell.
37. The method of claim 33, wherein the cell is a maize plant
cell.
38. The method of claim 33, wherein hydrolysis is detected by
spectrophotometry.
39. The method of claim 33, wherein hydrolysis is detected by
fluorescence.
40. The method of claim 33, wherein hydrolysis is detected by
phosphorescence.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/430,626 filed Dec. 3, 2002, which is herein
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to methods for
nucleic acid detection. More specifically, embodiments of the
invention relate to a polynucleotide that acts as a "marker" for
the presence of the same or a different polynucleotide of interest.
In such embodiments, methods rely on the use of organophosphate
hydrolase activity as a marker which has both selectable and
screenable properties.
[0004] 2. Description of the Related Art
[0005] Methods to detect nucleic acids and to detect specific
nucleic acids provide a foundation upon which the large and rapidly
growing field of molecular biology is built. There is constant need
for alternative methods and products. The reasons for selecting one
method over another are varied, and include a desire to avoid
radioactive materials, the lack of a license to use a technique,
the cost or availability of reagents or equipment, the desire to
minimize the time spent or the number of steps, the accuracy or
sensitivity for a certain application, the ease of analysis, or the
ability to automate the process.
[0006] The detection of nucleic acids or specific nucleic acids is
often a portion of a process rather than an end in itself. There
are many applications of the detection of nucleic acids in the art,
and new applications are always being developed. The ability to
detect and quantify nucleic acids is useful in detecting
microorganisms, viruses and biological molecules, and thus affects
many fields, including human and veterinary medicine, food
processing and environmental testing. Additionally, the detection
and/or quantification of specific biomolecules from biological
samples (e.g., tissue, sputum, urine, blood, semen, saliva) has
applications in forensic science, such as the identification and
exclusion of criminal suspects and paternity testing as well as in
genetics and medical diagnostics.
[0007] However, many attempts have been made to genetically
engineer desired traits into genomes by introduction of exogenous
genes using genetic engineering techniques. An important aspect of
the success achieved in genetic engineering has been the ability to
select or screen for transgenic cells. Most of the first successes
in genetic engineering relied on utilization of selectable markers
for identification of transgenic cells. Markers which have been
used for selection of transgenic cells include, for example, genes
that confer resistance to antibiotics and other-toxins, including,
for example, ampicillin, neomycin, puromycin, methotrexate or
tetracycline, those that complement auxotrophic deficiencies, or
those supply critical nutrients not available from complex media.
Other selectable markers include the dihydrofolate reductase gene,
which confers resistance to methotrexate, and thymidine kinase, or
genes conferring resistance to G418 or hygromycin. Commonly used
selectable markers for plant transformation include a neomycin
phosphotransferase gene (Potrykus et al., Mol. Gen. Genet. 199:183
(1985)), which provides resistance to kanamycin, paromomycin and
G418; a bar gene which codes for bialaphos or phosphinothricine
resistance (U.S. Pat. No. 5,550,318); a mutant aroA gene which
encodes an altered EPSP synthase protein conferring glyphosate
resistance (Hinchee et al., Bio/Technology 6:915-22 (1988)); a
nitrilase gene such as bxn from Klebsiella ozaenae which confers
resistance to bromoxynil (Stalker et al., J. Biol. Chem.
263:6310-14 (1988)); a mutant acetolactate synthase gene (ALS)
which confers resistance to imidazolinone, sulfonylurea or other
ALS inhibiting chemicals (European Patent Application No. 154,204,
1985); a methotrexate resistant DHFR gene (Thillet et al., J. Biol.
Chem. 263:12500-08 (1988)); a dalapon dehalogenase gene that
confers resistance to the herbicide dalapon; and a mutated
anthranilate synthase gene that confers resistance to 5-methyl
tryptophan.
[0008] More recently, interest has increased in utilization of
screenable or scorable markers. A screenable or scorable marker is
a gene that codes for a protein whose activity is easily detected,
allowing cells expressing such a marker to be readily identified.
Such screenable markers include a .beta.-glucuronidase, or uidA
gene (GUS), which encodes an enzyme for which various chromogenic
substrates are known; chloramphenicol acetyl transferase; alkaline
phosphatase; a R-locus gene, which encodes a product that regulates
the production of anthocyanin pigments (red color) in plant tissues
(Dellaporta et al., in CHROMOSOME STRUCTURE AND FUNCTION, Kluwer
Academic Publishers, Appels and Gustafson eds., pp. 263-282
(1988)); a p-lactamase gene (Sutcliffe, Proc. Nat'l. Acad. Sci.
U.S.A. 75:3737 (1978)), which encodes an enzyme for which various
chromogenic substrates are known (e.g., PADAC, a chromogenic
cephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad.
Sci. U.S.A. 80:1101 (1983)), which encodes a catechol dioxygenase
that can convert chromogenic catechols; an a-amylase gene (Ikuta et
al., Biotech. 8:241 (1990)); a tyrosinase gene (Katz et al., J.
Gen. Microbiol. 129:2703 (1983)), which encodes an enzyme capable
of oxidizing tyrosine to DOPA and dopaquinone, which in turn
condenses to form the easily detectable compound melanin; a
.beta.-galactosidase gene, which encodes an enzyme for which there
are chromogenic substrates; a lux gene, which encodes a luciferase,
the presence of which may be detected using, for example, X-ray
film, scintillation counting, fluorescent spectrophotometry,
low-light video cameras, photon counting cameras or multiwell
luminometry; and a green fluorescent protein (GFP) gene (Sheen et
al., Plant J. 8(5):777-84 (1995)).
[0009] Despite the abundance of selectable and screenable markers
for genetic engineering, there are very few, if any, markers which
have both selectable and screenable properties. It would be
beneficial if another marker were available for detecting the
presence of a polynucleotide. It is therefore an object of the
present invention to provide methods for determining whether a cell
has incorporated a polynucleotide using a marker which has
selectable and/or screenable properties.
SUMMARY OF THE INVENTION
[0010] The present invention provides novel methods for determining
whether a cell has incorporated a polynucleotide. Such a method
utilizes organophosphate hydrolase activity as a marker, which has
both selectable and screenable properties to detect the presence or
absence of the polynucleotide in the cell.
[0011] According to one aspect of the invention, there is provided
a method for determining whether a cell has incorporated and
expresses a polynucleotide comprising introducing to a cell a
construct comprising a polynucleotide encoding an enzyme having
organophosphate hydrolase activity; contacting the cell with an
organophosphate such that if the cell does not contain the
construct and an organophosphate hydrolase is not thereby produced,
the cell growth is inhibited, thereby determining whether the cell
has incorporated a polynucleotide.
[0012] According to another aspect of the invention, there is
provided a method for determining whether a cell has incorporated
and expresses a polynucleotide comprising introducing to a cell a
construct comprising a polynucleotide encoding an enzyme having
organophosphate hydrolase activity; contacting the cell with an
organophosphate such that if the cell contains the construct and an
organophosphate hydrolase is thereby expressed, the organophosphate
hydrolase hydrolyzes the organophosphate; and detecting the
hydrolysis, thereby determining whether the cell has incorporated a
polynucleotide.
[0013] According to yet another aspect of the invention, there is
provided a method for determining whether a cell has incorporated a
first polynucleotide comprising introducing to a cell a construct
comprising a first polynucleotide and a second polynucleotide,
wherein the second polynucleotide encodes an enzyme having
organophosphate hydrolase activity; contacting the cell with an
organophosphate such that if the cell does not contain the
construct and an organophosphate hydrolase is not thereby produced,
the cell growth is inhibited, thereby determining whether the cell
has incorporated a first polynucleotide.
[0014] According to yet another aspect of the invention, there is
provided a method for determining whether a cell has incorporated a
first polynucleotide comprising introducing to a cell a construct
comprising a first polynucleotide and a second polynucleotide,
wherein the second polynucleotide encodes an enzyme having
organophosphate hydrolase activity; contacting the cell with an
organophosphate such that if the cell contains the construct and an
organophosphate hydrolase is thereby expressed, the organophosphate
hydrolase hydrolyzes the organophosphate; and detecting the
hydrolysis, thereby determining whether the cell has incorporated a
first polynucleotide.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a graph showing the effect of the organophosphates
amiprophos-methyl, piperophos, bensulide, and coumaphos on maize
callus growth. Control callus growth was 824% of initial mass at
three weeks.
[0016] FIG. 2 shows the general scheme of hydrolysis of an
organophosphate by organophosphate hydrolase ("X" is 0, F, C, or S;
"R" is any alkyl group).
[0017] FIG. 3 shows the hydrolysis of amiprophos-methyl by
organophosphate hydrolase. A product of this reaction is
4-methyl-2-nitrophenol.
[0018] FIG. 4 is a spectrometer wavelength graph (scans taken every
30 seconds) in which cleavage of 0.5 mM of amiprophos-methyl by 2
.mu.g of organophosphate hydrolase results in the absorbance of
light. A product of the reaction, 4-methyl-2-nitrophenol, is yellow
in color (.epsilon.=1800 M.sup.-1cm.sup.-1 at 435 nm).
[0019] FIGS. 5A-C demonstrate the comparative effect of the
organophosphate bensulide on maize seedlings that have been
transformed with a polynucleotide encoding an enzyme having
organophosphate hydrolase activity ("OPA0403-2") and maize
seedlings that have not been so transformed ("elite line/HiII
Cross"). FIG. 5A shows the seedlings from the control group; FIG.
5B shows the seedlings following exposure to 0.35 ml of Bensumec
4LF; and FIG. 5C shows the seedlings following exposure to 3.5 ml
of Bensumec 4LF.
[0020] FIG. 6 is a haloxon graph showing the ability of transgenic
organophosphate hydrolase (OPH) cells to grow at concentrations
where control cultures would not.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides novel methods for determining
whether a cell has incorporated a polynucleotide. Such methods
utilize an enzyme that has organophosphate hydrolase activity. Such
hydrolysis can be detected and a polynucleotide encoding such an
enzyme can be used as a scorable and/or selectable marker to
determine whether the polynucleotide has been incorporated into a
cell.
[0022] As used herein, the term "organophosphate hydrolase
activity" is intended to refer to that activity which results in
the hydrolysis of an organophosphate.
[0023] Many cells contain very low levels of endogenous
organophosphate hydrolase activity that would not interfere with
the ability of the user of the inventive methods to determine if a
foreign polynucleotide was indeed incorporated into the cell, and
the inventors do not wish to be limited to the use of those cells
which contain no organophosphate hydrolase activity. Such
measurements of enzyme activity are readily known to those of skill
in this art.
[0024] Hydrolysis of Organophosphates by Organophosphate
Hydrolase
[0025] Synthetic organophosphates are used extensively as
agricultural and domestic pesticides, including insecticides,
fungicides, and herbicides. FIG. 1 demonstrates the inhibitory
effect that the organophosphate herbicides amiprophos-methyl,
piperophos, and bensulide, and the organophosphate insecticide
coumaphos, has on new growth from maize callus. Naturally occurring
bacterial isolates capable of metabolizing this class of compounds
have received considerable attention since they provide the
possibility of both environmental and in situ detoxification.
Pseudomonas putida MG, Pseudomonas diminuta, and Flavobacterium
spp. possess the ability to degrade an extremely broad spectrum of
organophosphate phosphotriesters as well as thiol esters. McDaniel
et al., J. Bacteriology 170(5):2306-11 (1988).
[0026] Organophosphate hydrolase (EC 3.1.8.1) is a broad spectrum
enzyme that is capable of detoxifying organophosphates by creating
various phosphoryl bonds (P--O, P--F, P--CN, and P--S) between the
phosphorous center and varying electrophilic leaving groups. Dave
et al., Chem.-Biol. Interact. 87:55-68 (1993). FIG. 2 demonstrates
the general scheme of hydrolysis of an organophosphate by
organophosphate hydrolase. This enzyme is often identified by other
names such as paraoxonase; A-esterase; aryltriphosphatase;
organophosphate esterase; esterase B1; esterase E4; paraoxon
esterase; pirimiphos-methyloxon esterase; OPA anhydrase;
organophosphorus hydrolase; phosphotriesterase; paraoxon hydrolase;
OPH; organophosphorus acid anhydrase; DFPase; parathion hydrolase;
parathion aryl esterase; somanase; and sarinase. The hydrolytic
reaction rates with several phosphotriesterases appear to be
limited by diffusion to the active center of the enzyme. Caldwell
et al., Biochemistry 30:7438-44 (1991). Organophosphate hydrolase
is the only enzyme which has been shown to be able to hydrolyze the
P--S bond of various phosphorothioate pesticides. The toxicity of
hydrolyzed products has been shown to be significantly reduced as
indicated by the loss of inhibition of acetylcholinesterase
activity and by decreased neurotoxic response in animals.
Kolakowski et al., Biocat. Biotrans. 15:297-312 (1997).
Phosphorothioate pesticides such as acephate, azinophos-methyl,
demeton-S-methyl, malathion, and phosalone have been shown to be
hydrolyzed by organophosphate hydrolase. The hydrolysis of these
pesticides has a first order dependency on the amount of enzyme
used and the reaction time. Organophosphate hydrolase hydrolyzes
acephate, azinophos-methyl, demeton-S-methyl and phosalone at rates
which are thousands of times greater than that which occurs during
strong alkaline hydrolysis. In contrast, the enzyme possesses poor
capability for malathion hydrolysis, although still significantly
better than non-enzymatic hydrolysis under similar conditions. When
compared to the hydrolysis of P--O bond phosphotriester substrates
and P--F bond phosphofluoridate substrates, the thioesters (P--S
bond esters) hydrolysis was much slower in general. See Kolakowski
et al., supra.
[0027] Other organophosphates that have been shown or have the
potential to be hydrolyzed by organophosphate hydrolase include,
but are not limited to, the microtubule assembly inhibitor
amiprophos-methyl, the cell division inhibitor piperophos, the
lipid synthesis inhibitor bensulide, paraoxon, DFP, coumaphos,
soman, and VX.
[0028] Hydrolysis of Amiprophos-Methyl by Organophosphate
Hydrolase
[0029] FIG. 3 demonstrates the scheme of hydrolysis of the
organophosphate amiprophos-methyl by organophosphate hydrolase. A
product of this hydrolytic reaction is the compound
4-methyl-2-nitrophenol. This compound is yellow in color
(.epsilon.=1800 M.sup.-1cm.sup.-1 at 435 nm), as demonstrated by
the spectrometer wavelength graph in FIG. 4.
[0030] Polynucleotide Encoding Enzyme Having Organophosphate
Hydrolase Activity
[0031] A polynucleotide encoding an enzyme having organophosphate
hydrolase activity is preferably obtained from Flavobacterium sp.
(Genbank accession numbers M29593 or M22863), but can also be
obtained from other sources, including, but not limited to,
Pseudomonas diminuta (Genbank no. M20392) and Agrobacterium
tumefaciens (Genbank no. AY043245).
[0032] Polynucleotides encoding an enzyme having organophosphate
hydrolase or organophosphate hydrolase-like activity, such as
paraoxonase (PON) and organophosphate acid anhydrase, are also
contemplated. PON hydrolyzes P--C, P--O, P--F, and P--CN bonds, but
not P--S bonds, and has been isolated from Homo sapiens (human;
Genbank no. NMA.sub.--000446), Mus musculus (mouse; Genbank no.
NM.sub.--008896), Gallus gallus (chicken; Genbank no. BM427017),
Orycolagus cuniculus (rabbit; Genbank no. AF220944), Danio rerio
(zebra fish; Genbank no. B1709981), Xenopus laevis (frog; Genbank
no. BE506006), Rattus norvegicus (rat; Genbank no. AA817964), and
Melagris gallopavo (prairie chicken; Genbank no. MA7572).
Organophosphate acid anyhdrase hydrolyzes P--O, P--F, and P--CN
bonds, and has been isolated from Mycobacterium sp. (Genbank no.
M91040) and Nocardia sp. (Genbank no. JC1378).
[0033] Polynucleotide Constructs, Vectors, and Cells
[0034] A construct comprising a polynucleotide encoding an enzyme
having orgnanophosphate hydrolase activity is one that permits
expression of the polynucleotide. Such a construct typically
further comprises a transcription unit of a promoter operably
linked to the polynucleotide which is functional in a cell, and a
termination or polyadenylation signal. The construct can further
comprise additional polynucleotides that do not encode an enzyme
having organophosphate hydrolase activity, however are expressed in
the cell.
[0035] The polynucleotide construct typically forms part of a
transformation vector. Generally, such vectors comprise cis-acting
control regions effective for expression in a cell operatively
linked to the polynucleotide to be expressed. Appropriate
trans-acting factors either are supplied by the cell, supplied by a
complementing vector or supplied by the vector itself upon
introduction to the cell.
[0036] A great variety of expression vectors can be used to express
a polynucleotide encoding an enzyme having organophosphate
hydrolase activity as well as other polynucleotides. Such vectors
include chromosomal, episomal and virus-derived vectors, e.g.,
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, from viruses such
as baculoviruses, papova viruses, such as SV40, vaccinia viruses,
adenoviruses, fowl pox viruses, pseudorabies viruses and
retroviruses, and vectors derived from combinations thereof, such
as those derived from plasmid and bacteriophage genetic elements,
such as cosmids and phagemids. Generally, any vector suitable to
maintain, propagate or express polynucleotides to express a
polypeptide in a cell can be used for expression in this
regard.
[0037] A polynucleotide encoding an enzyme having organophosphate
hydrolase activity contained in the vector is operatively linked to
appropriate expression control sequence(s), including, for
instance, a promoter to direct mRNA transcription. Representatives
of such promoters include the phage lambda PL promoter, the E. coli
lac, trp and tac promoters, the SV40 early and late promoters, and
promoters of retroviral LTRs, to name just a few of the well-known
promoters. For maize expression vectors, promoters include, for
example, the ubiquitin and globulin promoters. In general,
expression vectors will contain sites for transcription, initiation
and termination, and, in the transcribed region, a ribosome binding
site for translation. The coding portion of the mature transcripts
expressed by the vectors will include a translation initiating AUG
at the beginning and a termination codon (UAA, UGA or UAG)
appropriately positioned at the end of the polypeptide to be
translated.
[0038] In addition, the vectors can contain control regions that
regulate as well as engender expression. Generally, such regions
will operate by controlling transcription, such as repressor
binding sites and enhancers and leaders, among others.
[0039] The vector comprising a construct comprising a
polynucleotide encoding an enzyme having organophosphate hydrolase
activity, as well as an appropriate promoter, and other appropriate
control sequences, and optional additional polynucleotides, can be
introduced to an appropriate cell using a variety of well known
techniques suitable to expression therein of a polynucleotide
encoding an enzyme having organophosphate hydrolase activity.
Representative examples of appropriate cells include bacterial
cells, such as E. coli, Streptomyces and Salmonella typhimurium
cells; fungal cells, such as yeast cells; insect cells such as
Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO,
COS and Bowes melanoma cells; and plant cells, such as maize cells.
Cells for a great variety of expression constructs and vectors are
well known, and those of skill will be enabled by the present
disclosure readily to select a cell for expressing a polynucleotide
encoding an enzyme having organophosphate hydrolase activity in
accordance with the invention. Preferably, the cell is one that
does not naturally contain an enzyme having significant
organophosphate hydrolase activity.
[0040] Among vectors preferred for use in bacteria are pQE70, pQE60
and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and
pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors
are listed solely by way of illustration of the many commercially
available and well known vectors available to those of skill in the
art.
[0041] Selection of appropriate constructs, vectors, and promoters
for expression in a cell is a well known procedure, and the
requisite techniques for expression construct/vector construction,
introduction of a construct, or vector comprising a construct, to a
cell and expression in the cell are routine skills in the art.
[0042] Transformation
[0043] Once a polynucleotide encoding an enzyme having
organophosphate hydrolase activity is obtained, it is introduced to
an appropriate cell. Preferably, such introduction is accomplished
through the use of a vector. The vector can be, for example, a
plasmid vector, a single or double-stranded phage vector, or a
single or double-stranded RNA or DNA viral vector. Such vectors can
be introduced to cells as polynucleotides, preferably DNA, by
well-known techniques for introducing DNA and RNA to cells. Viral
vectors can be replication competent or replication defective. In
the latter case viral propagation generally will occur only in
complementing cells.
[0044] Introduction of a vector comprising a polynucleotide
encoding an enzyme having organophosphate hydrolase activity to a
cell can be effected by calcium phosphate transfection,
DEAE-dextran mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection or other
methods. Such methods are described in many standard laboratory
manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY,
Appleton & Lang, Norwalk, Conn. (1986).
[0045] For plant cells, various methods are known in the art to
accomplish genetic transformation. Among these methods are
Agrobacterium species transformation and direct gene transfer.
[0046] Agrobacterium tumefaciens is the etiologic agent of crown
gall. The wild type form of Agrobacterium tumefaciens carries the
Ti (tumor-inducing) plasmid that directs the production of
tumorigenic crown gall growth on the host plants. The crown gall is
produced following the transfer of the tumor inducing T-DNA region
from the Ti plasmid into the genome of an infected plant. This DNA
fragment encodes genes for auxin and cytokinin biosynthesis, and it
is these hormones in high concentration that promote growth of
undifferentiated cells in the crown gall. Transfer of the T-DNA to
the plant genome requires that the Ti plasmid-encoded virulence
genes as well as the T-DNA borders, a set of direct DNA repeats
that delineate the region to be transferred. The tumor inducing
genes can be removed from Ti plasmid vectors, disarming the
pathogenic nature of the system, without affecting the transfer of
DNA fragments between the T-DNA borders. Therefore, the tumor
inducing genes are generally replaced with a gene encoding
resistance to kanamycin, or some other gene, to allow for selection
of transformants, and a gene encoding the desired trait. The
Agrobacterium containing the engineered plasmid is co-cultivated
with cultured plant cells or wounded tissue. The de-differentiated
plant cells are then propagated on selective media, and a
transgenic plant is subsequently regenerated from the transformed
cells by altering the levels of auxin and cytokinin in the growth
medium.
[0047] Current protocols for Agrobacterium mediated transformation
often employ binary vector systems, which divide the Ti plasmid
into two components, a shuttle vector and a helper plasmid. The
helper plasmid, which is permanently placed in the Agrobacterium
host, carries the virulence genes. However, a much smaller shuttle
vector contains T-DNA borders, a broad-host range bacterial origin
of replication, antibiotic resistance markers, and a multiple
cloning site for incorporation of the foreign gene. In the
alternative, a similar strategy employs cointegrating Ti plasmid
vectors, whereby an intermediate plasmid containing antibiotic
resistance, the gene to be transferred and one T-DNA border are
used to transform A. tumefaciens containing a disarmed Ti plasmid
possessing the virulence genes and one T-DNA border. The two
plasmids homologously recombined in vivo at the T-DNA borders
placing the antibiotic resistance gene and the gene of interest
between two T-DNA borders, one from each plasmid. The genes are
then transferred into plant tissue upon co-cultivation.
[0048] The Agrobacterium system has been well studied and has
further been developed into a system which permits routine
transformation of a variety of plant tissues. See, e.g., Schell et
al., Bio/Technology 1:175 (1983); Chilton, Scientific American
248:50 (1983). Some of the tissues transformed utilizing
Agrobacterium include tobacco, Barton et al., Cell 32:1033 (1983);
tomato, Fillatti et al., Bio/Technology 5:726 (1987); sunflower,
Everett et al., Bio/Technology 5:1201 (1987); cotton, Umbeck et
al., Bio/Technology 5:263 (1987); canola, Pua et al.,
Bio/Technology 5:815 (1987); potato, Facciotti et al.,
Bio/Technology 3:241 (1985); poplar, Pythoud et al., Bio/Technology
5:1323 (1987); and soybean, Hinchee et al., Bio/Technology 6:915
(1988).
[0049] In a preferred method of Agrobacterium transformation, the
Agrobacterium transformation methods described in U.S. Pat. No.
5,591,616 are generally followed, with modifications that the
inventors have found improve the number of transformants obtained.
This method uses the A188 variety of maize that produces Type I
callus in culture. In one preferred embodiment the HiII maize line
is used which initiates Type II embryogenic callus in culture.
While selection on phosphinothricin is recommended when using the
bar or PAT gene for selection, another preferred embodiment
provides for use of bialaphos instead.
[0050] The bacterial strain used in this protocol is LBA4404 with
the 40 kb super binary plasmid containing three vir loci from the
hypervirulent A281 strain. The plasmid has resistance to
tetracycline. The cloning vector cointegrates with the super binary
plasmid. Since the cloning vector has an E. coli specific
replication origin, but not an Agrobacterium specific replication
origin, it cannot survive in Agrobacterium without cointegrating
with the super binary plasmid. Since the LBA4404 strain is not
highly virulent, and has limited application without the super
binary plasmid, the inventors have found in yet another embodiment
that the EHA101 strain is preferred. It is a disarmed helper strain
derived from the hypervirulent A281 strain. The cointegrated super
binary/cloning vector from the LBA4404 parent is isolated and
electroporated into EHA 101, selecting for spectinomycin
resistance. The plasmid is isolated to assure that the EHA101
contains the plasmid.
[0051] Further, the protocol as described provides for growing a
fresh culture of the Agrobacterium on plates, scraping the bacteria
from the plates, and resuspending in the co-culture medium, as
stated in U.S. Pat. No. 5,591,616 for incubation with the maize
embryos. This medium includes 4.3 g MS salts, 0.5 mg nicotinic
acid, 0.5 mg pyridoxine hydrochloride, 1.0 ml thiamine
hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 g sucrose, and 36
g glucose, all at a pH of 5.8. In a further preferred method, the
bacteria are grown overnight in a 1 ml culture, and then a fresh 10
ml culture re-inoculated the next day when transformation is to
occur. The bacteria grow into log phase, and are harvested at a
density of no more than OD.sub.600=0.5 and is preferably between
0.2 and 0.5. The bacteria are then centrifuged to remove the media
and resuspended in the co-culture medium. Since HiII is used,
medium preferred for HiII is used. This medium is described in
considerable detail by Armstrong et al., Planta 154:207-14 (1985).
The resuspension medium is the same as that described above. All
further HiII media are as described in Armstrong et al., supra. The
result is redifferentiation of the plant cells and regeneration
into a plant. Redifferentiation is sometimes referred to as
dedifferentiation, but the former term more accurately describes
the process where the cell begins with a form and identity, is
placed on a medium in which it loses that identity, and becomes
"reprogrammed" to have a new identity. Thus the scutellum cells
become embryogenic callus.
[0052] Another vector for biological plant transformation is
Agrobacterium rhizogenes. A. rhizogenes, which incite root
formation in many dicotyledonous plant species, carries the Ri
(root-inducing) plasmid which functions in a manner analogous to
the Ti plasmid of A. tumefaciens. Transformation using A.
rhizogenes has also been successfully utilized to transform plants,
for example, alfalfa, Sukhapinda et al., Plant Mol. Biol. 8:209
(1987); Solanum nigrum L., Wei et al., Plant Cell Reports 5:93
(1986); and, poplar, Pythoud et al., supra.
[0053] Several direct gene transfer procedures have also been
developed to transform plant cells. In the direct transformation of
protoplasts, the uptake of exogenous genetic material into a
protoplast can be enhanced by use of a chemical agent or electric
field. The exogenous material can then be integrated into the
nuclear genome. Microprojectile bombardment, electroporation in
addition to several other direct transformation methods exist and
are known to those of skill in the art.
[0054] Electroporation is another effective means of introducing
foreign DNA to plant cells. Saul et al., "Direct DNA Transfer to
Protoplasts With and Without Electroporation," PLANT MOLECULAR
BIOLOGY MANUAL, vol. A1, Kluwer Academic Publishers, Dordrecht, The
Netherlands, Gelvin and Schilperoort eds., p. 1 (1988). Since any
DNA fragment can be delivered to the cell, this technique has the
advantage of allowing assimilation of a gene without having to
clone the DNA into a host vector such as A. tumefaciens.
[0055] Another technique for delivering DNA to intact plant tissue
or protoplasts is biollistic projection or microprojectile
bombardment. Tomes et al., Plant Mol. Biol. 14:21 (1990); Svab et
al., Proc. Nat'l. Acad. Sci. U.S.A. 87:8526-30 (1990). With this
technique, microprojectile particles (e.g., 1.2 micrometer gold or
Tungsten beads) coated with DNA are accelerated at high speeds into
plant tissue. This approach has the distinct advantage of being
applicable to any intact plant tissue or region of the plant and
has been used to transform organellar DNA. Svab et al., supra;
Boynton et al., Science 240:534 (1988). This technique is
especially useful for plants that are otherwise recalcitrant with
respect to transformation and/or regeneration. For instance,
biollistic methods have been successfully used to produce
transgenic monocot cereal plants. Potrykus (1991), supra; Wu et
al., "Transformation and Regeneration of Important Crop Plants;
Rice is the Model System for Monocots," in GENE MANIPULATION AND
PLANT IMPROVEMENT, vol. 2, Gustafson, J. P., ed., Quantum Press,
New York, p. 251 (1990); Gordon-Kamm et al., Plant Cell 2:603
(1990).
[0056] DNA viruses have been used as gene vectors. Both Cauliflower
Mosaic Virus (CaMV) and the closely related Figwort Virus are
circular duplex DNA viruses which replicate via transcription of a
full-length (35S) genomic RNA intermediate. A cauliflower mosaic
virus carrying a modified bacterial methotrexate-resistance gene
was used to infect a plant, whereby the foreign gene was
systemically spread in the plant. Brisson et al. Nature 310:511
(1984); Brisson et al., METHODS FOR PLANT MOLECULAR BIOLOGY,
Academic Press, San Diego, Weissbach and Weissbach eds., p. 437
(1988). The strong promoter responsible for the genomic replication
of the CaMV virus (the 35S promoter) has been extensively exploited
for the expression of heterologous genes in plants. Other
advantages of this system are the ease of infection, systemically
spread within the plant, and multiple copies of the gene per
cell.
[0057] Exogenous DNA can be introduced to cells or protoplasts by
microinjection. Microinjection is a method where a solution of
plasmid DNA is injected directly into the cell with a finely pulled
glass needle. Alfalfa protoplasts have been transformed utilizing
this method with a variety of plasmids. Reich et al.,
Bio/Technology 4:1001 (1986).
[0058] In liposome fusion, protoplasts and liposomes carrying the
foreign gene of interest are brought together. As membranes merge,
the foreign gene is transferred to the protoplast. Deshayes et al.,
EMBO J. 4:2731 (1985).
[0059] A form of chemical mediated transformation utilizes
polyethylene glycol (PEG) and has been carried out in N. tabacum a
dicot, and Lolium multiflorum, a monocot. It is a chemical
procedure of direct gene transfer based on synergistic interaction
between Mg.sup.2+, PEG, and possibly Ca.sup.2+. Negrutiu et al.,
Plant Mol. Biol. 8:363 (1987).
[0060] Detection of Hydrolysis of Organophosphate
[0061] Detection of the hydrolysis of an organophosphate by an
enzyme having organophosphate hydrolase activity will depend upon
the specific organophosphate being subjected to hydrolysis. For
example, hydrolytic reactions may proceed with the absorbance of
light (i.e., hydrolysis of amiprophos-methyl), which can be
detected by spectrophotometry. Additionally, hydrolytic reactions
may result in the production of compounds capable of emitting
light, for example, by fluorescence or phosphorescence (i.e.,
hydrolysis of coumaphos results in the production of a fluorescent
compound). Such methods are well known to those of skill in the
art. By way of illustration only, OPH activity, for example, can be
analyzed by the hydrolysis of paraoxon. Cleavage of paraoxon yields
p-Nitrophenol, which is measured spectrophotometrically at 400 nm.
So, OPH activity can be assayed in 1 ml plastic cuvettes by
observing the hydrolysis of Paraoxon to p-Nitrophenol at 400 nm.
Units of enzyme are determined using the extinction coefficient of
p-Nitrophenol (17 mM.sup.-1 cm.sup.-1).
[0062] In the foregoing discussion, a number of citations from
professional journals and patents are included for reference. All
such citations are hereby incorporated in their entirety by
reference.
EXAMPLE 1
[0063] Polynucleotide Encoding Enzyme Having Organophosphate
Hydrolase Activity
[0064] A polynucleotide encoding an enzyme having organophosphate
hydrolase activity was obtained from Flavobacterium sp. (Genbank
accession number M29593). The sequence was translated into a
protein sequence and then back translated into a DNA sequence using
a maize codon usage table with the BACKTRANSLATE program of the GCG
Wisconsin package (Wisconsin Package, version 9, Genetics Computer
Group (GCG), Madison, Wis.) and then searched for putative
deleterious sequences using the FINDPATTERN program of the GCG
Wisconsin package. Alternative codons were chosen to eliminate the
deleterious mRNA signals in the coding sequence and to add
convenient enzyme restriction sites to facilitate downstream
cloning. Codons that reflected less than 20% usage were avoided.
The completed sequence was analyzed for unique restriction sites
with the Vector NTI program. Five roughly equidistant sites were
chosen for the construction of the polynucleotide encoding the
enzyme having organophosphate hydrolase activity. Oligonucleotides
were ordered in 50 bp lengths with 25 bp overhangs. These were
annealed and amplified by PCR. Amplified products were trapped in a
vector and transformed into competent cells. Colonies were analyzed
by restriction analysis and by DNA sequencing. Correct clones were
then subcloned together in the vector. After the complete sequence
(SEQ ID NO:1) was assembled, a barley .alpha.-amylase signal
sequence (BAASS) (SEQ ID NO:2) was added to ensure high expression
in plants, and it was cloned into a maize expression vector under
the direction of the ubiquitin promoter and the pinII
terminator.
EXAMPLE 2
[0065] Transformation
[0066] A maize expression vector containing the ubiquitin promoter,
the BAASS (SEQ ID NO:2), the polynucleotide encoding an enzyme
having organophosphate hydrolase activity (SEQ ID NO:1), and the
pinII terminator was transferred to Agrobacterium by the method
described in U.S. Pat. No. 5,591,616, with modifications that
improve the number of transformants obtained. As previously
described, this method uses the A188 variety of maize that produces
Type I callus in culture. In one preferred embodiment the HiII
maize line is used which initiates Type II embryogenic callus in
culture. While selection on phosphinothricin is recommended when
using the bar or PAT gene for selection, another preferred
embodiment provides for use of bialaphos instead.
[0067] The bacterial strain used in this protocol is LBA4404 with
the 40 kb super binary plasmid containing three vir loci from the
hypervirulent A281 strain. The plasmid has resistance to
tetracycline. The cloning vector cointegrates with the super binary
plasmid. Since the cloning vector has an E. coli specific
replication origin, but not an Agrobacterium specific replication
origin, it cannot survive in Agrobacterium without cointegrating
with the super binary plasmid. Since the LBA4404 strain is not
highly virulent, and has limited application without the super
binary plasmid, the inventors have found in yet another embodiment
that the EHA101 strain is preferred. It is a disarmed helper strain
derived from the hypervirulent A281 strain. The cointegrated super
binary/cloning vector from the LBA4404 parent is isolated and
electroporated into EHA 101, selecting for spectinomycin
resistance. The plasmid is isolated to assure that the EHA101
contains the plasmid.
[0068] Further, the protocol as described provides for growing a
fresh culture of the Agrobacterium on plates, scraping the bacteria
from the plates, and resuspending in the co-culture medium, as
stated in U.S. Pat. No. 5,591,616 for incubation with the maize
embryos. This medium includes 4.3 g MS salts, 0.5 mg nicotinic
acid, 0.5 mg pyridoxine hydrochloride, 1.0 ml thiamine
hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 g sucrose, and 36
g glucose, all at a pH of 5.8. In a further preferred method, the
bacteria are grown overnight in a 1 ml culture, and then a fresh 10
ml culture re-inoculated the next day when transformation is to
occur. The bacteria grow into log phase, and are harvested at a
density of no more than OD.sub.600=0.5 and is preferably between
0.2 and 0.5. The bacteria are then centrifuged to remove the media
and resuspended in the co-culture medium. Since HiII is used,
medium preferred for HiII is used. This medium is described in
considerable detail by Armstrong et al., Planta 154:207-14 (1985).
The resuspension medium is the same as that described above. All
further HiII media are as described in Armstrong et al., supra. The
result is redifferentiation of the plant cells and regeneration
into a plant. Redifferentiation is sometimes referred to as
dedifferentiation, but the former term more accurately describes
the process where the cell begins with a form and identity, is
placed on a medium in which it loses that identity, and becomes
"reprogrammed" to have a new identity. Thus the scutellum cells
become embryogenic callus.
EXAMPLE 3
[0069] Maize Plant Regeneration from Type II Callus
[0070] Regeneration of plants from Type II callus is based upon
allowing the embryoids on the surface of the Type II callus to
mature and germinate. See Freeling & Walbot, The Maize Handbook
(1994) at pp. 673-674. The callus are first collected and weighed
in petri plates. 1-2 grams fresh weight of soft, friable Type II
callus containing numerous embryoids are evenly distributed over
the surface of a 100.times.15 mm petri plate which contains 25 ml
of regeneration medium. Regeneration medium consists of Murashige
and Skoog (MS) basal salts, modified White's vitamins (0.2 g/l
glycine, and 0.5 g/l of each of thiamine-HCl, pyridoxine-HCl, and
nicotinic acid), supplemented with 6% sucrose, 0.1 g/l
myo-inositol, and 0.8% Bacto-agar (6SMSOD). The plates are then
wrapped with Parafilm and placed in the dark. After one week, the
plates are moved to a lighted growth chamber with a 16-hour (75
.mu.E m.sup.-2sec.sup.-1) and an 8-hour dark photo period. Three
weeks after plating the Type II callus to the 6SMSOD, the plate is
examined for shoot formation from the calli. The calli and the
shoot are then transferred to fresh Bacto-agar plates for another
two weeks. The callus without shoots can be left on the Bacto-agar
for a longer period if the callus is slow in embryo development.
Upon distinct formation of a shoot and root (some may be ready for
transfer after the first three weeks on regeneration medium), the
newly developed green plantlets are transferred to Magenta GA-7
(Magenta Corp., Chicago, Ill.) containers with 60 ml of 3SMSOD
medium solidified with 0.6% Bacto-agar. When the plant has
developed a strong root system (10-15 days after transfer into
Magenta boxes), the plant is gently removed from the agar, the
remaining agar is washed from the roots and shoot, and the plant is
carefully transplanted into a 4-inch pot containing moist soil. The
pots can then be placed in a high humidity chamber and, over a
period of ten days, the humidity can be slowly reduced to
approximately that of the greenhouse. Once plants are adapted to a
lower humidity, they may be removed from the greenhouse and treated
like seedlings.
EXAMPLE 4
[0071] Use of Organophosphate Hydrolase as Selectable Marker in
Plants
[0072] Transformed and non-transformed seedlings were sprayed with
0.35 ml and 3.5 ml of Bensumec 4LF (PBI/Gordon Corp., Kansas City,
Mo.), the active ingredient of which is the organophosphate
bensulide, and examined for germination. FIGS. 5A-C show,
respectively, a control group of organophosphate
hydrolase-transformed ("OPA0403-2") and non-transformed ("elite
line/HiII Cross") seedlings that were not subjected to bensulide
treatment; transformed and non-transformed seedlings that were
subjected to 0.35 ml of Bensumec 4LF; and transformed and
non-transformed seedlings that were subjected to 3.5 ml of Bensumec
4LF. As demonstrated by FIG. 5B, the transformed seedlings
subjected to 0.35 ml of Bensumec 4LF exhibited greater resistance
to the organophosphate than the non-transformed seedlings. This
difference was even more apparent when the transformed and
non-transformed seedlings were subjected to 3.5 ml of Bensumec 4LF,
where, as shown in FIG. 5C, nearly all of the non-transformed
seedlings were killed, while the transformed seedlings were largely
unaffected.
EXAMPLE 5
[0073] Use of Organophosphate Hydrolase as Screenable Marker in
Plants
[0074] Two standard, 100.times.25 mm plant tissue culture plates
were prepared, each plate containing 35 ml of 563O medium. A
one-liter solution of 563O medium (pH 5.6 to 5.8) consists of N6
salts (4 g), Eriksson's vitamins (1 ml of 1000.times. stock),
thiamine-HCl (1 ml of 0.5 mg/ml stock), L-proline (0.7 g), sucrose
(20 g), MES buffer (0.5 g), 2,4-D (1.5 ml of 1 mg/ml stock), 8 g
agar, bialophos (1 ml of 1.6 mg/ml stock), silver nitrate (1 ml of
0.85 mg/ml stock), and carbenicillin (1 ml of 100 mg/ml stock).
[0075] Following plate preparation, 3 ml of a 1 mg/ml solution of
the organophosphate coumaphos (Chemservice #F2058) in ethanol was
spread onto the surface each plate with a glass plate spreader. The
plates were then placed in a laminar flow hood until the ethanol
evaporated. Once the plates were dry, maize callus transformed with
a maize expression vector containing the ubiquitin promoter, the
BAASS (SEQ ID NO:2), the polynucleotide encoding an enzyme having
organophosphate hydrolase activity (SEQ ID NO: 1), and the pinII
terminator ("transformed callus") was transferred to the surface of
one plate, and callus that was not transformed with a
polynucleotide encoding an enzyme having organophosphate hydrolase
activity ("non-transformed callus") was transferred to the surface
of the other plate. The plates were then sealed, and stored in a
standard growth chamber.
[0076] At two days post-transfer, the plates were examined under
ultraviolet light, and fluorescence, if any, was determined. Callus
on the plate containing transformed callus fluoresced, while callus
on the plate containing the non-transformed callus did not. Similar
results were observed at six days post-transfer.
[0077] These results indicate that the organophosphate coumaphos is
degraded/hydrolyzed by an enzyme having organophosphate hydrolase
activity, that such degradation/hydrolysis can be detected, and
that a polynucleotide encoding such an enzyme can be used as a
screenable marker for transformation.
EXAMPLE 6
[0078] Use of Organophosphate Hydrolase as Selectable Marker in
Non-Plants
[0079] Because organophosphates have insecticidal and fungicidal
properties in addition to herbicidal properties, it will be
appreciated by those of skill in the art that the cells used in the
inventive methods are not limited to plant cells. Accordingly, a
polynucleotide encoding an enzyme having organophosphate hydrolase
activity can be obtained from, for example, Flavobacterium sp.
(Genbank accession number M29593), and this sequence can be
translated into a protein sequence and then back translated into a
DNA sequence using, for example, an animal (i.e., insect) or fungal
(i.e., yeast) codon usage table with the BACKTRANSLATE program of
the GCG Wisconsin package (Wisconsin Package, version 9, Genetics
Computer Group (GCG), Madison, Wis.). The sequence can then be
searched for putative deleterious sequences using the FINDPATTERN
program of the GCG Wisconsin package. Alternative codons can be
chosen to eliminate the deleterious mRNA signals in the coding
sequence and to add convenient enzyme restriction sites to
facilitate downstream cloning. Codons that reflect less than 20%
usage can be avoided. The completed sequence can be analyzed for
unique restriction sites with the Vector NTI program. Five roughly
equidistant sites can be chosen for the construction of the
polynucleotide encoding the enzyme having organophosphate hydrolase
activity. Oligonucleotides can be ordered in 50 bp lengths with 25
bp overhangs. These can be annealed and amplified by PCR. Amplified
products can be trapped in a vector and transformed into competent
cells. Colonies can be analyzed by restriction analysis and by DNA
sequencing. Correct clones can be subcloned together in an
appropriate vector. After the complete sequence is assembled, a
suitable signal sequence can be added to ensure high expression in
the particular animal or fungal cell that is ultimately to be
transformed. The sequence can then be cloned into a suitable
expression vector having appropriate regulatory elements, and the
appropriate animal or fungal cells can be transformed using any of
variety of techniques known in the art for transforming such
cells.
[0080] Transformants can be selected for by contacting the cells
with an organophosphate such that if the cells do not contain the
polynucleotide encoding an enzyme having organophosphate hydrolase
activity and such an enzyme is not thereby produced, the cell
growth is inhibited.
EXAMPLE 7
[0081] Use of Organophosphate Hydrolase as Screenable Marker in
Non-Plants
[0082] A polynucleotide encoding an enzyme having organophosphate
hydrolase activity can be obtained from, for example,
Flavobacterium sp. (Genbank accession number M29593), and this
sequence can be translated into a protein sequence and then back
translated into a DNA sequence using, for example, an animal (i.e.,
insect), bacterial (i.e., E. coli) or fungal (i.e., yeast) codon
usage table with the BACKTRANSLATE program of the GCG Wisconsin
package (Wisconsin Package, version 9, Genetics Computer Group
(GCG), Madison, Wis.). The sequence can then be searched for
putative deleterious sequences using the FINDPATTERN program of the
GCG Wisconsin package. Alternative codons can be chosen to
eliminate the deleterious mRNA signals in the coding sequence and
to add convenient enzyme restriction sites to facilitate downstream
cloning. Codons that reflect less than 20% usage can be avoided.
The completed sequence can be analyzed for unique restriction sites
with the Vector NTI program. Five roughly equidistant sites can be
chosen for the construction of the polynucleotide encoding the
enzyme having organophosphate hydrolase activity. Oligonucleotides
can be ordered in 50 bp lengths with 25 bp overhangs. These can be
annealed and amplified by PCR. Amplified products can be trapped in
a vector and transformed into competent cells. Colonies can be
analyzed by restriction analysis and by DNA sequencing. Correct
clones can be subcloned together in an appropriate vector. After
the complete sequence is assembled, a suitable signal sequence can
be added to ensure high expression in the particular animal,
bacterial, or fungal cell that is ultimately to be transformed. The
sequence can then be cloned into a suitable expression vector
having appropriate regulatory elements, and the appropriate animal,
bacterial, or fungal cells can be transformed using any of variety
of techniques known in the art for transforming such cells.
[0083] Transformants can be selected for by contacting the cells
with an organophosphate such that if the cells contain the
polynucleotide encoding an enzyme having organophosphate hydrolase
activity and such an enzyme is thereby produced, the
organophosphate is hydrolyzed. Such hydrolysis can be detected by
suitable means.
EXAMPLE 8
[0084] Production of Enzyme having Organophosphate Hydrolase
Activity
[0085] An enzyme having organophosphate hydrolase activity that is
produced by transformed cells containing a polynucleotide encoding
an enzyme having organophosphate hydrolase activity is purified by
standard methods known to those in the art.
PROSPECTIVE EXAMPLE 9
[0086] Use of Organophosphate Hydrolase as a Selectable Marker in
Cell Cultures
[0087] This prospective example shows how to evaluate control cells
for their ability to grow in the presence of organophosphates and
to evaluate transgenic cultures for their ability to grow in the
presence of a selected organophosphate.
[0088] A concentration is selected that allows for the cells to
grow when organophosphate hydrolase (OPH) is expressed but not when
it is absence in the cell cultures. By way of prospective example,
haloxon can be made up in 100, 10 and 1 mg/ml stocks in DMSO. These
stock solutions can be used to make plant tissue cultures plates
with haloxon concentrations of 1, 5, 10, 50, 100, and 500 .mu.M.
Control plates contain a similar amount of DMSO compared to the
haloxon plates. The tissue culture is a modified 5630 media without
added bialaphos. Approximately 0.5 g of callus tissue is added to
each plate. Exact tissue mass is calculated by pre-weighing the
plate prior to the addition of callus and weighing the plate after
the tissue has been added. The control cells are transformed with
the OPH gene. Such transformation methods are well known in the
art. Tissue is transferred to fresh media about every two weeks for
six weeks and the tissue mass is calculated at every plate change.
Then select for cells growing in the presence of the selected
organophosphate and regenerate plants. The plants can then be
verified as transgenic by a variety of methods, such as enzymatic
active of OPH, PCR of the gene, scorable active of the OPH, or the
presence of a second gene that was co-transformed with OPH. In
order to compare both lines, results are reported as percentage
growth compared to the control. The total growth of each line at
each treatment is divided by the total growth of the control over
the same period and multiplied by 100. This percentage is then
graphed. Control results are graphed at 0.1 on the .mu.M Haloxon
axis due to the logarithmic scale. The haloxon graph as shown in
FIG. 6 demonstrates the ability of the transgenic OPH cells to grow
at concentrations where control cultures would not.
[0089] One of ordinary skill in the art, with the aid of the
present disclosure, can affect various changes, substitutions of
equivalents and other alterations to the methods and compositions
herein set forth, in order to practice this invention. Therefore,
the protection granted by letters patent should not be limited
except by the language of the claims as set forth below.
Sequence CWU 1
1
2 1 982 DNA Artificial Sequence Sequence originally obtained from
Flavobacterium sp., Genbank accession number M29593. Sequence
translated and back-translated with BACKTRANSLATE (Wisc. GCG, ver.
9). Deleterious sequences removed with FINDPATTERN (Wisc. GCG, ver.
9). 1 cggcccgatc accatctccg aggccggctt caccctcacc cacgagcaca
tctgcggctc 60 ctccgccggc ttcctccgcg cctggccgga gttcttcggc
tcccgcaagg ccctcgccga 120 gaaggccgtg cgcggcctcc gccgcgcccg
cgccgccggc gtgcgcacca tcgtggacgt 180 gtccaccttc gacatcggcc
gcgacgtgtc cctcctcgcc gaggtgtccc gcgccgccga 240 cgtgcacatc
gtggccgcca ccggcctctg gttcgacccg ccgctctcca tgcgcctccg 300
ctccgtggag gagctcaccc agttcttcct ccgcgagatc cagtacggca tcgaggacac
360 cggcatccgc gccggcatca tcaaggtggc caccaccggc aaggccaccc
cgttccagga 420 gctcgtgctc aaggccgccg cccgcgcctc cctcgccacc
ggcgtgccgg tgaccaccca 480 caccgccgcc tcccagcgcg acggcgagca
gcaggccgcc atcttcgagt ccgagggcct 540 ctccccgtcc cgcgtgtgca
tcggccactc cgacgacacc gacgacctct cctacctcac 600 cgccctcgcc
gcccgcggct acctcatcgg cctcgaccac atcccgcact ccgccatcgg 660
cctcgaggac aacgcctccg cgtccgccct cctcggcatc cgctcctggc agacccgcgc
720 cctcctcatc aaggccctca tcgaccaggg ctacatgaag cagatcctcg
tgtccaacga 780 ctggctcttc ggcttctcct cctacgtgac caacatcatg
gacgtgatgg accgcgtgaa 840 cccggacggc atggccttca tcccgctccg
cgtgatcccg ttcctccgcg agaagggcgt 900 gccgcaggag accctcgccg
gcatcaccgt gaccaacccg gcccgcttcc tctccccgac 960 cctccgcgcc
tcctgagtta ac 982 2 100 DNA Artificial Sequence barley
alpha-amylase signal sequence (BAASS) 2 ccatggccaa caagcacctg
agcctctccc tcttcctcgt gctcctcggc ctctccgcct 60 ccctcgccag
cggcaccggc gaccgcatca acaccgtgcg 100
* * * * *